This invention relates to the identification of a class of nuclease inhibitors, e.g., ribonulcease (RNase) inhibitors. The inhibitors bind to and inhibit nucleases, particularly RNases. The invention provides methods for removing these inhibitors from buffers and other reagents employed in research to generate purified buffers and reagents useful for applications involving RNase and other nucleases. The invention further provides methods for inhibiting RNase and other nucleases using one or more of the inhibitors identified. The invention also relates to methods and materials for removing undesired RNase and other nucleases from biological media.
Biochemical research and biotechnology rely on polymeric nucleic acids. Yet during their storage and use, nucleic acids encounter nucleases, both advertently and inadvertently. For example, nucleases are often added with the intent of destroying RNA in a DNA sample, or vice versa. Residual amounts of these nucleases can affect downstream steps in protocols. (Pasloske, B. L. In Nuclease Methods and Protocols, Schein, C. H., Ed. Humana Press: Totowa, N.J., 2001; pp 105-111; Sweeney, R. Y.; Kelemen, B. R.; Woycechowsky, K. J.; Raines, R. T. Anal. Biochem. 2000, 286, (2), 312-314). Alternatively, human skin is an abundant source of nucleases that can be transferred accidentally to surfaces and solutions (Holley, R. W.; Apgar, J.; Merrill, S. H. J. Biol. Chem. 1961, 236, PC42-43.) Moreover, reagents (including those labeled “nuclease free”) are often contaminated with nucleases (Hengen, P. N. Trends Biochem. Sci. 1996, 21, (3), 112-113).
RNA is the least stable of the biopolymers that effect information transfer in biology (Wolfenden, R., and Snider, M. J. (2001) Acc. Chem. Res. 34, 938-945). The lifetime of RNA in vivo is most often determined by measuring the rate of its enzymatic degradation (Ross, J. (1996) Trends Genet. 12, 171-175). Ribonucleases are perhaps the most problematic of nucleases because of their high natural abundance, prodigious catalytic activity, notorious conformational stability and resistance to proteolysis, and lack of requisite cofactors. (D'Alessio, G.; Riordan, J. F., Ed., Ribonucleases: Structures and Functions. Academic Press: New York, 1997; Raines, R. T. Chem. Rev. 1998, 98, 1045-1065).
In vitro, ribonuclease inhibitors are often employed to mitigate damage to RNA from incidental (or inadvertent) contamination with secretory ribonucleases such as the human homolog of ribonuclease A (RNase A, 1 EC 3.1.27.5) (Raines, R. T. (1998) supra). The abundance and diversity of natural ribonucleases has led to an ever-increasing interest in inhibitor design and discovery (Russo, A., Acharya, K. R., and Shapiro, R. (2001) Methods Enzymol. 341, 629-648). Although several ribonuclease inhibitors have been described, each suffers from one or more undesirable attribute (See: Pasloske (2001) supra; Raines (1998) supra; Russo, A.; Acharya, K. R.; Shapiro, R. Methods Enzymol. 2001, 341, 629-648.) For example, the ribonuclease inhibitor protein (RI) binds ribonucleases with femtomolar affinity (Hofsteenge, J. In Ribonucleases: Structures and Functions, D'Alessio, G.; Riordan, J. F., Ed. Academic Press: New York, 1997; pp 621-658; Shapiro, R. Methods Enzymol 2001, 341, 611-628), but is expensive and highly sensitive to oxidation (Kim, B.-M.; Schultz, L. W.; Raines, R. T. Protein Sci. 1999, 8, 430-434). In addition, RI inhibits only ribonuclease A (RNase A6, 11; EC 3.1.27.5) and some of its homologs. Although diethylpyrocarbonate (DEPC) inactivates many nucleases, it is toxic and its use requires time-consuming procedures. Moreover, DEPC-treatment results in the covalent modification of many proteins, nucleic acids, and small molecules (Miles, E. W. Methods Enzymol. 1977, 47, 431-442).
In vitro, ribonuclease inhibitors are useful in a variety of molecular biology applications where RNase contamination is a potential problem. Examples of these applications include RNA isolation, purification and storage, particularly mRNA isolation and purification, assays characterizing or employing RNA, reverse transcription of mRNA, cell-free translation systems, preparation of RNase-free antibodies, reverse transcription-PCR and in vitro virus replication.
Ideally, ribonuclease inhibitors to be used in these kinds of applications will be capable of inhibiting a large number of different RNases, such as eukaryotic RNase A, RNase B and RNase C, as well as prokaryotic RNases. Thus, there is a need in the art for ribonuclease inhibitors for various applications.
We have recently reported that MES—NaOH buffer (pH 6.0) inhibits catalysis by RNase A and a variant thereof designated K7A/R10A/K66A at low salt concentrations (Park and Raines (2000) FEBS Lett. 468, 199-202). The inhibitor was described in this reference as “highly charged,” anionic and not polymeric. The origin of RNase inhibition in the buffer was described as “low levels of small anions in common buffer solution.” The K7A/R10A/K66A RNase A variant has three fewer cationic residues than does the wild-type enzyme [Fisher, B. M., Ha, J.-H. and Raines, R. T. (1998) Biochemistry 37, 12121-12132; Fisher, B. M., Schultz, L. W. and Raines, R. T. (1998) Biochemistry 37, 17386-17401] and is effectively missing two of the four known subsites for phosphoryl group binding [Fisher, B. M., Grilley, J. E. and Raines, R. T. (1998) J. Biol. Chem. 273, 34134-34138; Nogues, M. V., Moussaoui, M., Boix, E., Vilanova, M., Ribo, M. and Cuchillo, C. M. (1998) Cell. Mol. Life. Sci. 54, 766-774].
This invention is based at least in part on the identification of the inhibitor(s) in MES—NaOH (2-(N-morpholino)ethane sulfonate-NaOH) buffer as relatively low molecular weight oligomers of vinyl sulfonic acid, e.g.:
where n represents the number of monomers. Smith et al. (2003) J. Biol. Chem., which is incorporated by reference herein to the extent that it is not inconsistent with the disclosure herein, provides details of the identification of the inhibitors.
Various low molecular weight RNase inhibitors, including nucleosides and nucleotides, have been identified (Richards and Wyckoff, 1971; Sambrook, J. et al. (1982) “Molecular Cloning, A Laboratory Manual” Cold Spring Harbor Laboratory and later editions). Uridine-vanadate (UV) is a particularly potent RNase inhibitor with Ki=10:M at pH 7 (Linguist, R. N. et al. (1973) J. Amer. Chem. Soc. 95:8762-8768; Wlodawer, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80:3628-3631.)
Ribonucleoside vanadyl complex mixture is a commercial product (Sigma-Aldrich, described as inhibiting approximately 2×104 Kunitz units/ml of RNase A at 20 mM) used as a ribonuclease inhibitor during cell lysis and mRNA purification (Berger, S. L. and Birkenmeier, C. S. (1979) Biochemistry 18:5145). U.S. Pat. No. 5,852,001 reports RNase inhibitors that are nucleotides having diphosphate groups.
Additional reported ribonulcease inhibitors include certain clays (e.g., bentonite and macaloid), certain surfactants (e.g., SDS, EDTA); proteinase K and ammonium sulfate (see: Jocoli and Ronald (1973) Can. J. Biochem, 51:1558-1565; Jones (1976) Biochem Biophys Res. Commun, 69:469-474; Mendelsohn and Young Biochem. Biophys. Acta (1978) 519:461-473; Allewell and Sama (1974) Biochem. Biophys. Acta, 341-484-488).
The 50-kD ribonuclease inhibitor protein (R1) forms a tight 1:1 complex with RNase A (Kd˜10−14) (Lee et al., 1989) chelating all of its phosphoryl group binding subsites (Kobe and Deisenhofer, 1996.) The utility of pyrophosphate linked oligonucleotides and ribonuclease inhibitor is limited, both by the difficulty and expense of their production and by their intrinsic instability (Russo and Shapiro, 1999; Kim, et al., 1999)
U.S. Pat. No. 6,664,379 reports methods and compositions for inhibiting nucleases using anti-nuclease antibodies.
Certain polyanions are reported to be effective inhibitors of RNase A (Richards and Wyckoff, 1971). Heparin, tyrosine-glutamate copolymers, and a number of different polysulfates and polyphosphates are reported to inhibit catalysis by the enzyme (Sela, 1962; Zollner and Fellig, 1953; Heymann et al., 1958).
Poly(vinylsulfonic acid) (or poly(vinylsulfonate) (PVS), also called poly-ethenesulfonic acid, PES) is reported to inhibit RNase activity at pH 7.6 (M. K Bach, 1964). PVS samples having molecular weight ranging from 5,700 (g/mol) to 27,600 (g/mol) were assessed for inhibition of RNase at various concentrations. Molecular weights were reported to be estimated by a combination of light scattering, viscosimetric analysis and ultracentrifugation essentially as in Dailer and Kerber (1955) Makromol. Chem. 17:56. Percent inhibition of RNase was reported to generally increase with increasing molecular weight of the PVS and increasing amount of the PVS present. The results presented for the lowest molecular weight PVS assessed do not exhibit consistent inhibition. PVS of molecular weight 5,700 exhibited inhibition at an intermediate concentration, but not at lower or higher concentrations. PVS of molecular weight 6400 exhibited stimulation of RNase at lower concentrations and inhibition of RNase at the higher concentration tested. Results reported for inhibition of RNase by PVS of molecular weight 12,900 also appear inconsistent with the general trend of the data presented. The reference suggests that only longer polymers with molecular weight greater than 9,000 were good inhibitors of RNase.
PVS is also reported to be a potent inhibitor of DNase (Tunis M. and Regelson W. Arch. Biochem. Biophys. 1963 101, 448-455; Bach (1964) supra).
Fellig and Wiley, 1959 reported that polyvinyl sulfonate was a “fairly effective” inhibitor of ribonuclease (pancreatic) compared to sulfated polyvinyl alcohol which was described as “a very strong inhibitor.” Inhibition by polyvinyl sulfonate was reported to decrease markedly with increasing sodium chloride concentration. The molecular weight of the polyvinyl sulfonate employed in these experiments was not reported.
In contrast, Littauer and Sela, 1962 reported that PVS (having reported molecular weight of 300,000) exhibited no significant activity on crude ribosomal E. Coli RNase at pH 7.4.
Cheng et al., 1974 report that their isolation of spleen mRNA was carried out in the presence of an RNase inhibitor, such as polyvinylsulfate or bentonite. The molecular weight and source of the polyvinylsulfate used was not reported. Mach et al., 1968 employed polyvinyl sulfonic acid in a cell-free system for active protein synthesis. The reference, however, does not report the function of the polyvinylsulfonic acid used and the source and molecular weight of the material is not given.
Niehaus W. G. and Flynn T. (1993) Mycopathologia 123(3): 155-158 reported that a contaminant of MES buffer inhibited a number of fungal NADP-dependent dehydrogenases. The inhibitor was identified as an “ethylenesulfonic acid oligomer” and it was suggested to be a “model compound” for the development of an antifungal agent. It was reported that the MES buffer contained large polymers (˜50,000 g/mol) of ethylenesulfonic acid (i.e., polyvinyl sulfonate.) In a later publication, (Niehaus W. G. et al. (1995) Arch. Biochem. Biophys. 324(2):325-330), it was reported that polyvinyl sulfonate (Mr (relative molecular weight)=50,000) was a potent inhibitor of a number of fungal enzymes.
Japanese published patent application 05139981 A (published Jun. 8, 1993) Abstract (Eng) reports that the sodium salt of vinylsulfonic acid polymer (PVS), as well as several other sulfonated polymers, function as antiviral agents, particularly against HIV. These agents are described as having “inhibitory activity for cytoclasis due to human immunodeficiency virus, inhibitory activity for giant cell formation and anti-human immunodeficiency virus activity such as reverse transcriptase inhibitory activity.”
R. Kisilevsky et al., 2002 reports that short-chain aliphatic polysulfonates inhibit the entry of Pasmodium into red blood cells and may be useful as antimalarial agents. Two samples of poly(vinylsulfonate sodium salt) were employed which were “mixtures of oligomeric species with slightly different chain lengths and stereochemistry.” The first (designated compound 1) was prepared from commercial poly(vinylsulfonate sodium salt) (Aldrich Company, catalog no. 27, 842-4, a 25% (wt/wt) solution in water) as follows:                (The) solution (2 liters) was concentrated under reduced pressure to half of its volume. Ethanol (200 ml) was added, followed by the addition of activated carbon (50 g). The mixture was warmed on a steam bath for 20 min and then filtered through Celite, and the solvent was removed under reduced pressure to give a light-yellowish oil. The oil was dried in a vacuum oven (50° C.) for 3 days to give an amorphous solid (486 g).        
The molecular weight distribution was determined using gel permeation chromatography by a commercial laboratory (American Polymer Standards) and reported as follows: number-average molecular weight (Mn)=1,800; weight-molecular weight (Mw))=3,050; z-average molecular weight (Mz)=5,400; Mw/Mn (polydispersity index)=1.69.
The second sample of poly(vinylsulfonate sodium salt) (designated compound 2) was prepared by radical polymerization of sodium vinylsulfonate in the presence of sodium persulfate. Commercially available sodium vinylsulfonate (Aldrich, catalog no. 27, 841-6) as a 25% (wt/wt) solution in water was employed. The sodium vinylsulfonate solution was diluted in water 160 ml of the 25% (wt/wt) solution in (160 ml water) and purged with argon for 30 min. A solution of sodium persulfate (1.6 g) in H2O (30 ml) was similarly purged. The purged solutions were mixed and the mixture was heated (under argon) at 80° C. in an oil bath for 17 h. The reaction mixture was concentrated under reduced pressure to 100 ml; addition of methanol (1 liter) gave a white solid which was dried in a vacuum oven (75° C.) overnight to give compound 2. The molecular weight distribution of compound 2 as determined by gel permeation chromatography (American Polymer Standards Corporation) was reported as follows: Mn=1,600; Mw=2,000; Mz=2,700; and Mw/Mn=1.25.
Compound 1 is reported to have an IC50 of 0.2 μM (on the basis of the weight molecular weight of 3,050) for Plasmodium falciparum viability in human red blood cells. Compound 2 is reported to have an IC50 of 6±2 μM (on the basis of a weight molecular weight of 2,000) in the same assay. The poly vinylsulfonate sample (compound 1) having higher weight molecular weight was found to be more effective against P. Falciparum. Aliphatic polysulfonates were said to be potent inhibitors of merozoite invasion of red blood cells and to possibly constitute a novel class of antimalarials. It is suggested that compounds 1 and 2, of the reference, are inhibiting the initial step of the invasion process, possibly by interacting with a merozite surface protein or with a red blood cell receptor.
Kisilevsky et al. (1995) Nature Medicine 1(2):143-148 reports experiments on arresting amyloidosis in vivo using small-molecule anionic sulfonates and sulfates. Poly (vinylsulfonate sodium salt) as a 25% (wt/wt) solution (Aldrich, 27, 842-4) “processed to provide an amorphous solid” said to have a molecular weight distribution of 900-1,000 was reported to interfere with heparin sulfate-stimulated ∃-peptide fibril aggregation in vitro and to substantially reduce murine splenic AA amyloid progression in vivo. The method by which PVS samples were processed prior to use was not provided.